Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli

An amylase gene was identified in a Streptococcus bovis 033 gtWESB genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.     

Cloning and expression of an alpha-acetolactate decarboxylase gene from Streptococcus lactis subsp. diacetylactis in Escherichia coli.

The Streptococcus lactis gene coding for alpha-acetolactate decarboxylase (ADC) was cloned in Escherichia coli. Subsequent subcloning in E. coli showed that the ADC gene was located within a 1.3-kilobase DNA fragment. The ADC gene was controlled by its own promoter. Gas chromatography showed that S. lactis and the transformed E. coli strains produced the two optical isomers of acetoin in different ratios.     

Cloning and expression of an alpha-acetolactate decarboxylase gene from Streptococcus lactis subsp. diacetylactis in Escherichia coli

The Streptococcus lactis gene coding for alpha-acetolactate decarboxylase (ADC) was cloned in Escherichia coli. Subsequent subcloning in E. coli showed that the ADC gene was located within a 1.3-kilobase DNA fragment. The ADC gene was controlled by its own promoter. Gas chromatography showed that S. lactis and the transformed E. coli strains produced the two optical isomers of acetoin in different ratios.     

Cloning and expression of the transhydrogenase gene of Escherichia coli.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.     

Cloning of a tyrosinase gene in Streptococcus thermophilus

Summary The streptococcal cloning vector pIL253 (4.96-kbp, Em^r) was used to introduce the Streptomyces antibioticus tyrosinase (mel) gene (1.56-kbp) into S. thermophilus, an important microbe in dairy fermentations. Electrotransformants of S. thermophilus ST128 contained 6.51-kbp recombinant plasmids which probed positively in Southern hybridizations with the biotin-labeled mel fragment. Western blots of cell extracts resolved by SDS-PAGE showed the presence of a ca. 31-kDa band thus confirming the synthesis of tyrosinase protein by genetic transformants.     

Cloning and expression of the metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli.

The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322. A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R. albus. The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R. albus. The size of the cloned fragment was reduced by using HindIII and EcoRI. The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1). Cellulase production by E. coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens.

An endoglucanase gene of Ruminococcus flavefaciens FD1 was cloned on the vector pEcoR251 to form the recombinant plasmid pMEB200. The cloned endoglucanase gene showed carboxymethylcellulase enzyme activity but no degradation of Avicel (FMC Corp., Philadelphia, Pa.) or filter paper. Carboxymethylcellulase activity was found during the late-exponential-growth phase and accumulated in the periplasmic fraction. Enzyme production was not subject to catabolite repression by glucose.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

D-乳酸高产菌菊糖芽胞乳杆菌Y2-8磷酸果糖激酶基因在大肠杆菌中的克隆和表达

以D-乳酸高产菌菊糖芽胞乳杆菌Y2-8基因组DNA为模板,通过PCR扩增得到960 bp的磷酸果糖激酶基因(pfk)。氨基酸序列比对分析表明,该磷酸果糖激酶(PFK)与其他乳酸菌PFK具有保守的底物结合位点,但是其变构效应物结合位点存在差异。将pfk基因克隆到表达载体pSE380上,获得重组菌E-pSE-pfk。进一步通过诱导条件的优化,重组菌的PFK比酶活达到4.89 U/mg,是优化前的4.79倍。采用低温诱导策略有助于实现菊糖芽胞乳杆菌pfk基因在大肠杆菌中可溶性表达。     

Cloning the gene of beta-galactosidase from the industrial strain of Streptococcus lactis 111 in E. coli cells and conjugated transfer of this gene to Streptococcus thermophilus cells

The ability of the industrial strains of Streptococcus lactis to synthesize the enzyme beta-galactosidase was studied. Five strains among sixteen were found to produce high levels of the enzyme. The beta-galactosidase gene in the most active strain Streptococcus lactis 111 was shown to be located on the 50 kb conjugative plasmid. The plasmid was transferred by conjugation into Streptococcus thermophilus cells and subsequently the gene for beta-galactosidase was studied in transconjugants. The beta-galactosidase gene from Streptococcus lactis 111 was subcloned in Escherichia coli cells on the plasmid pBR322. The gene was localized on the 4.8 kb BgIII fragment of DNA. Following the restriction of DNA by the Sau3A the gene was subcloned on the birepliconed plasmid vector pCB20 capable of replication in the Gram-negative as well as Gram-positive microorganisms. The recombinant derivatives of pCB20 were isolated that carry the beta-galactosidase gene on the DNA fragments of different size.     

Cloning and expression of the Escherichia coli K-12 sad gene.

The Escherichia coli K-12 sad gene, which encodes an NAD-dependent succinic semialdehyde dehydrogenase, was cloned into a high-copy-number vector. Minicells carrying a sad+ plasmid produced a 55,000-dalton peptide, the probable sad gene product.     

Cloning and expression of the pepD gene of Escherichia coli

Peptidase D of Escherichia coli, cleaving the unusual dipeptide carnosine, was found to be encoded by the ColE1 hybrid plasmid pLC44-11. From this plasmid the pepD gene was subcloned into small vectors. As shown by successive reduction of the flanking sequences of genomic DNA, the order of genes in the region at 6 min of the E. coli K12 map is phoE, pepD, in the clockwise orientation. Insertional inactivation of the pepD gene and expression of recombinant plasmids in maxicells allowed the identification of the pepD product as a 52 kDa protein. Comparison with the 100 kDa protein molecular mass determined by gel filtration suggests that active peptidase D is probably a dimer.     

Cloning and expression in Escherichia coli of a xylanase-encoding gene from the yeast Cryptococcus albidus.

In the yeast, Cryptococcus albidus, a comparison between the sequence of the xylanase (XLN)-encoding chromosomal gene (XLN) and the cDNA sequence reveals the presence of seven introns, ranging in length from 51 to 69 bp. One of their 5' splice site sequences is similar to the consensus sequence for yeast, while the other six resemble the consensus sequence for higher eukaryotes. Their 3' end splice site sequences are representative of the conserved sequence found in eukaryotes. Their putative branching point sequences are different from the well-known conserved sequence, 5'-TACTAAC, observed in yeast, but again resemble the mammalian one. The cDNA encoding XLN is expressed by Escherichia coli, under the control of the lacZ promoter. The gene product remains inside the cell and has a molecular size of 40 kDa, which matches the size of the nonglycosylated protein. When compared to the glycosylated enzyme, the nonglycosylated XLN from E. coli shows twofold less affinity for substrate and its Vmax is 100-fold lower. Moreover, the nonglycosylated XLN only acts on large xylan polymers and very slightly on xylohexaose.     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23.

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.